Improved volume CLEM revealed that aberrant phagophores and RB1CC1/FIP200-containing clusters appear surround SQSTM1/p62 aggregates in Atg9a-deficient cells
ATG9A is an important membrane protein in mammalian macroautophagy. The formation of autophagosomes and phagophores is blocked in atg9a KO cells. However, it remains possible that residual membrane formation activity exists in these cells. These precursor structures that precede phagophores are, if...
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Taylor & Francis Group
2023-12-01
|
| Series: | Autophagy Reports |
| Subjects: | |
| Online Access: | http://dx.doi.org/10.1080/27694127.2023.2256599 |
| _version_ | 1797641008300687360 |
|---|---|
| author | Soichiro Kakuta Junji Yamaguchi Chigure Suzuki Isei Tanida Yasuo Uchiyama |
| author_facet | Soichiro Kakuta Junji Yamaguchi Chigure Suzuki Isei Tanida Yasuo Uchiyama |
| author_sort | Soichiro Kakuta |
| collection | DOAJ |
| description | ATG9A is an important membrane protein in mammalian macroautophagy. The formation of autophagosomes and phagophores is blocked in atg9a KO cells. However, it remains possible that residual membrane formation activity exists in these cells. These precursor structures that precede phagophores are, if they exist, rare and may be difficult to find. Here, we introduce the modified volume correlative light and electron microscopy (CLEM) method to analyze these structures three-dimensionally. In addition to target proteins, mitochondria were labeled as a landmark for precise correlation of slice images by a confocal fluorescence microscope and a focused ion beam scanning electron microscope. We found phagophores and small membrane vesicles near SQSTM1/p62 aggregates in atg9a KO cells, indicating that phagophores could be formed in atg9a-deficient cells, although they were immature and inefficient. Furthermore, we found that RB1CC1/FIP200-positive structures formed clusters around SQSTM1/p62 with ferritin and TAX1BP1. Taken together, our method contributes to the understanding of undiscovered fine structures. Abbreviations: CLEM: correlative light and electron microscopy; EM: electron microscopy; ER: endoplasmic reticulum; FIB-SEM: focused ion beam scanning electron microscopy; FM: fluorescence microscopy; GFP: green fluorescent protein; KO: knock out; MEF: mouse embryonic fibroblast; PBS: phosphate-buffered saline; ROI: region of interest; SEM: scanning electron microscopy. |
| first_indexed | 2024-03-11T13:39:24Z |
| format | Article |
| id | doaj.art-6e574d7da9f64470a356e2e4c3d30b16 |
| institution | Directory Open Access Journal |
| issn | 2769-4127 |
| language | English |
| last_indexed | 2024-03-11T13:39:24Z |
| publishDate | 2023-12-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | Autophagy Reports |
| spelling | doaj.art-6e574d7da9f64470a356e2e4c3d30b162023-11-02T13:48:32ZengTaylor & Francis GroupAutophagy Reports2769-41272023-12-012110.1080/27694127.2023.22565992256599Improved volume CLEM revealed that aberrant phagophores and RB1CC1/FIP200-containing clusters appear surround SQSTM1/p62 aggregates in Atg9a-deficient cellsSoichiro Kakuta0Junji Yamaguchi1Chigure Suzuki2Isei Tanida3Yasuo Uchiyama4Laboratory of Morphology and Image Analysis, Biomedical Research Core Facilities, Juntendo University Graduate School of MedicineLaboratory of Morphology and Image Analysis, Biomedical Research Core Facilities, Juntendo University Graduate School of MedicineJuntendo University Graduate School of MedicineJuntendo University Graduate School of MedicineJuntendo University Graduate School of MedicineATG9A is an important membrane protein in mammalian macroautophagy. The formation of autophagosomes and phagophores is blocked in atg9a KO cells. However, it remains possible that residual membrane formation activity exists in these cells. These precursor structures that precede phagophores are, if they exist, rare and may be difficult to find. Here, we introduce the modified volume correlative light and electron microscopy (CLEM) method to analyze these structures three-dimensionally. In addition to target proteins, mitochondria were labeled as a landmark for precise correlation of slice images by a confocal fluorescence microscope and a focused ion beam scanning electron microscope. We found phagophores and small membrane vesicles near SQSTM1/p62 aggregates in atg9a KO cells, indicating that phagophores could be formed in atg9a-deficient cells, although they were immature and inefficient. Furthermore, we found that RB1CC1/FIP200-positive structures formed clusters around SQSTM1/p62 with ferritin and TAX1BP1. Taken together, our method contributes to the understanding of undiscovered fine structures. Abbreviations: CLEM: correlative light and electron microscopy; EM: electron microscopy; ER: endoplasmic reticulum; FIB-SEM: focused ion beam scanning electron microscopy; FM: fluorescence microscopy; GFP: green fluorescent protein; KO: knock out; MEF: mouse embryonic fibroblast; PBS: phosphate-buffered saline; ROI: region of interest; SEM: scanning electron microscopy.http://dx.doi.org/10.1080/27694127.2023.22565993d-clemautophagosomeautophagyelectron microscopyfib-semisolation membrane |
| spellingShingle | Soichiro Kakuta Junji Yamaguchi Chigure Suzuki Isei Tanida Yasuo Uchiyama Improved volume CLEM revealed that aberrant phagophores and RB1CC1/FIP200-containing clusters appear surround SQSTM1/p62 aggregates in Atg9a-deficient cells Autophagy Reports 3d-clem autophagosome autophagy electron microscopy fib-sem isolation membrane |
| title | Improved volume CLEM revealed that aberrant phagophores and RB1CC1/FIP200-containing clusters appear surround SQSTM1/p62 aggregates in Atg9a-deficient cells |
| title_full | Improved volume CLEM revealed that aberrant phagophores and RB1CC1/FIP200-containing clusters appear surround SQSTM1/p62 aggregates in Atg9a-deficient cells |
| title_fullStr | Improved volume CLEM revealed that aberrant phagophores and RB1CC1/FIP200-containing clusters appear surround SQSTM1/p62 aggregates in Atg9a-deficient cells |
| title_full_unstemmed | Improved volume CLEM revealed that aberrant phagophores and RB1CC1/FIP200-containing clusters appear surround SQSTM1/p62 aggregates in Atg9a-deficient cells |
| title_short | Improved volume CLEM revealed that aberrant phagophores and RB1CC1/FIP200-containing clusters appear surround SQSTM1/p62 aggregates in Atg9a-deficient cells |
| title_sort | improved volume clem revealed that aberrant phagophores and rb1cc1 fip200 containing clusters appear surround sqstm1 p62 aggregates in atg9a deficient cells |
| topic | 3d-clem autophagosome autophagy electron microscopy fib-sem isolation membrane |
| url | http://dx.doi.org/10.1080/27694127.2023.2256599 |
| work_keys_str_mv | AT soichirokakuta improvedvolumeclemrevealedthataberrantphagophoresandrb1cc1fip200containingclustersappearsurroundsqstm1p62aggregatesinatg9adeficientcells AT junjiyamaguchi improvedvolumeclemrevealedthataberrantphagophoresandrb1cc1fip200containingclustersappearsurroundsqstm1p62aggregatesinatg9adeficientcells AT chiguresuzuki improvedvolumeclemrevealedthataberrantphagophoresandrb1cc1fip200containingclustersappearsurroundsqstm1p62aggregatesinatg9adeficientcells AT iseitanida improvedvolumeclemrevealedthataberrantphagophoresandrb1cc1fip200containingclustersappearsurroundsqstm1p62aggregatesinatg9adeficientcells AT yasuouchiyama improvedvolumeclemrevealedthataberrantphagophoresandrb1cc1fip200containingclustersappearsurroundsqstm1p62aggregatesinatg9adeficientcells |